Evidence for surface-coat localisation of a monoclonal antibody-isolated merozoite antigen of Babesia divergens.

The localisation of monoclonal antibody-derived merozoite antigens of Babesia divergens was examined using immunogold electron microscopy and immunoprecipitation of the monoclonal antibody with both biosynthetically and surface-labelled parasites. Immunogold labelling provided evidence that the antigens are components of the surface coat of the merozoite. Immunoprecipitation with biosynthetically labelled parasites showed the antigens to be of parasite origin, whereas surface labelling confirmed that the antigens form part of the surface coat.

A monoclonal antibody-derived antigen of Babesia divergens: characterization and investigation of its ability to protect gerbils against virulent homologous challenge.

A Babesia divergens merozoite antigen was purified by affinity chromatography using a monoclonal antibody. Silver staining of SDS-PAGE gels revealed 2 bands of Mr 50-60 kDa and Mr 24-29. It is proposed that the Mr 24-29 kDa band represents the native protein and that it is complexed, perhaps as a dimer or perhaps to host protein to give the Mr 50-60 kDa band. Peptide analysis of the antigen followed by Western blotting with the monoclonal antibody revealed only an Mr 20-24 kDa band. After affinity purification the antigen was precipitated with acetone and inoculated subcutaneously, together with complete Freund's adjuvant, into gerbils. Two inoculations were given 3 weeks apart, after which the immunized animals and a group of control animals were challenged with 5 x 10(7) parasitized red cells from a virulent microaerophilus stationary phase (MASP) culture of an homologous strain of B. divergens. Parasitaemias rose rapidly in control animals and all were dead by the fifth day. Two of the vaccinated animals survived the challenge. Parasitaemias in the remaining two rose slowly but both animals succumbed on the seventh day. Western blotting of merozoites and of the purified antigen, using sera from the surviving animals showed strong reactivity with the Mr 24-29 kDa band.

A strain of Babesia divergens, attenuated after long term culture.

The Weybridge strain of Babesia divergens became less virulent after 18 months in culture and was believed to be attenuated. Inoculations with the attenuated line using as many as 5 x 10(8) infected erythrocytes failed to raise a normal infection in Mongolian gerbils (Meriones unguiculatus) whereas, with the line that had been passaged from culture through gerbils at bimonthly intervals, inoculations of 10(7) infected erythrocytes gave rise to fatal infections. Gerbils were immunised with the attenuated line using doses ranging from 5 x 10(5) to 5 x 10(7) infected erythrocytes. Parasitaemias in the infected gerbils did not exceed 0.7 per cent after immunisation. All animals were protected when challenged with the virulent line four weeks later. Control animals died four days after challenge infection.

Induction of protective immunity to Babesia divergens in Mongolian gerbils, Meriones unguiculatus, using culture-derived immunogens.

Immunogens derived from microaerophilous stationary phase (MASP) cultures of Babesia divergens grown in bovine erythrocytes were used to inoculate the laboratory host of B. divergens, the Mongolian gerbil, Meriones unguiculatus. Animals inoculated subcutaneously twice with preparations of freeze-thawed merozoites in complete Freund's adjuvant were fully protected against homologous challenge, as were gerbils immunised with a non-viable preparation of parasite-enriched lysed infected bovine erythrocytes. Animals which had been infected with small numbers of parasitised erythrocytes from cultures cooled to 4 degrees C, allowed to recover, then challenged, also survived. All three groups had high antibody titres which dropped immediately after challenge and then rose again. Gerbils given culture supernatants containing soluble merozoite protein coat antigens were partially protected only after receiving a third inoculation. Non-immunised animals all died 4 days after challenge.

A monoclonal antibody to Babesia divergens which inhibits merozoite invasion.

A monoclonal antibody-producing hybridoma cell line has been raised against merozoites of Babesia divergens. This antibody is strongly reactive against merozoites in an enzyme-linked immunosorbent assay (ELISA) and is also positive in an indirect fluorescent antibody test (IFAT) on preparations of live merozoites. The antibody recognizes an antigen, of molecular weight (Mr) 50-60 K by Western blot analysis on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Merozoite neutralization assays show that the antibody significantly inhibits merozoite invasion of bovine erythrocytes.

Antigen-specific suppression of human antibody responses by allogeneic T cells. III. Role of the major histocompatibility complex.

Specific antibody responses obtained in vitro from human blood mononuclear cells (PBM) were profoundly suppressed by allogeneic T cells. Experiments carried out with combinations of cells from HLA identical siblings, and HLA identical but unrelated donors, showed that suppression depended upon HLA incompatibility between responding PBM and allogeneic Ts. In order to map the specific HLA loci concerned, a series of experiments were undertaken using combinations of cells from a large number of HLA typed donors. Significant suppression was found to occur in every combination of HLA incompatible cells tested, including those with nonidentity at HLA-A, B, DR, A and DR, or B and DR, suggesting that suppression can be generated by nonidentity at class I or class II loci. With some HLA-A homozygous donors, however, a dominant role for class I (HLA-A) antigens was indicated by the finding of one-directional suppression in combinations where the HLA-A locus was seen as foreign by one partner only (A3,----A2,3; and A2----A2,26). Similar one-directional suppression was also seen with cells from a pair of siblings who were HLA identical except for a single A locus antigen arising from an HLA-A/B recombination (A3,----A3,1). These results indicate an important, but not exclusive role for class I MHC antigens in the activation of allogeneic Ts. The way in which this occurs is unknown, but one possibility is that it results from the activation of normal antigen-specific Ts by the interaction of their receptors for self-MHC with cross-reacting alloantigens.

Antigen-specific suppression of human antibody responses by allogeneic T cells. II. Cell interactions involved in the generation of suppression.

Specific antibody responses to influenza virus were obtained in vitro from human blood mononuclear cells (PBMC). Antibody production in these cultures was profoundly suppressed by the addition of allogeneic T cells with the surface phenotype Leu2a+ (CD8+), Leu8-. Suppression by allogeneic T suppressor (Ts) cells required interactions only between T-depleted B (E-) cells and allogeneic Leu2a+. No evidence was obtained for T-T cell interactions, or for Ts inducer cells similar to those described for nonspecific antibody responses to pokeweed mitogen. Moreover, allogeneic E+, or allogeneic Leu2a+ cells were able to suppress specific antibody responses by E- cells when help was provided by T cell-replacing factor showing that the target of suppression was the responding E- cells, and not T helper cells. In contrast to allogeneic T cells, allogeneic E- cells did not suppress antibody production when added to cultures of unfractionated PBMC (E- + E+). That is, Ts cells activated to allogeneic E- were unable to suppress antibody production by the syngeneic E- cells present in the same culture tube. This result shows that alloactivated Ts cells were specific for the allogeneic E- target cells, and that suppression was not mediated by nonspecific allogeneic effects. Allogeneic Ts cells therefore differ from Ts cells in pokeweed mitogen responses by their specificity, and by their activation in the absence of Ts inducer cells.

Specificity repertoire of lymphocytes from multiple myeloma patients. I. High frequency of B cells specific for idiotypic and F(ab')2-region determinants on immunoglobulin.

The specificity repertoire of B lymphocytes from 14 multiple myeloma patients has been studied using the technique of Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) coupled with clonal analysis by limiting dilution. We find that up to 100% of the B cells from myeloma patients undergoing EBV transformation secrete IgM specific for determinants on the F(ab')2 region of autologous and/or heterologous monoclonal immunoglobulin. In normal individuals 0.02-0.73% of the transformed B cells secrete IgM specific for F(ab')2 determinants. Two patients with monoclonal gammopathy of undetermined significance had only a weak reactivity to F(ab')2 fragments. The number of anti-F(ab')2 B cells was up to 145-fold greater in patients than in normal donors. The majority of antibodies from patient clones recognized determinants shared among 3-12 different F(ab')2 fragments, whereas those originating from normal donor B cells saw determinants expressed on only one or two of the panel of test F(ab')2 fragments. There was a preference for autologous M components and a high proportion of antiidiotypic reactivity in five of eight patients so analyzed. We speculate that these findings indicate the existence of an anti-F(ab')2 immunoregulatory network mediating patient immunodeficiency network mediating patient immunodeficiency, thereby creating an abnormality that may enable the progression of multiple myeloma.

Antigen-specific suppression of human antibody responses by allogeneic T cells. I. Frequency and antigen specificity of allogeneic suppressor T cells and their role in major histocompatibility complex-controlled genetic restriction.

Specific antibody responses to influenza virus were obtained in vitro from human blood mononuclear cells (PBM). The addition of allogeneic T cells to responding PBM profoundly suppressed antigen-induced antibody responses, but had no effect on pokeweed mitogen (PWM)-induced polyclonal Ig formation. This raised the possibility that suppression by allogeneic T cells may result from the activation of antigen-specific T suppressor (Ts) cells rather than nonspecific allogeneic effects. The frequency of allogeneic Ts in PBM from a number of different donors, estimated in a series of limiting dilution analyses, was found to range from 0.8 X 10(-5) to 4.5 X 10(-5), which is more typical of antigen-specific than alloreactive T cells. By adding limiting numbers of allogeneic T cells to antibody-forming cultures stimulated simultaneously with two non-cross-reacting antigens (influenza A and B strain viruses A/X31 and B/HK), it was possible to demonstrate suppression of the response to one antigen, but not the other, in the same culture well. Moreover, the frequency of wells in which the response to both antigens was suppressed was not significantly different from that predicted from the calculated frequency of specific allogeneic Ts. These results show that allogeneic suppression was antigen specific, and was not due to non-specific allogeneic effects. By separating T cell preparations into Leu-3a+ (helper) and Leu-2a+ (suppressor/cytotoxic) T cell subsets, suppression was shown to be mediated by a radiosensitive Leu-2a+ T cell. The removal of Leu-2a+ cells from T cell preparations abrogated the suppressor effect and permitted T cell collaboration with B cells, across an HLA-A, -B, and -DR barrier. This result shows that in at least some combinations, suppression rather than major histocompatibility complex restriction is the reason for the failure of allogeneic T and B cells to collaborate in T cell-dependent antibody responses.

Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization.

This paper describes a simple protocol for the efficient generation of large numbers of human monoclonal antibody-producing cells. This system is based on initial limiting-dilution culture after Epstein-Barr virus exposure of highly enriched precursors selected from peripheral blood mononuclear cells. Precursors can be enriched by using rosetting or panning approaches. Antibodies to erythrocytes, a mouse mammary carcinoma, DNA, and sperm antigens, produced without any deliberate immunization, are described. Large-scale human monoclonal antibody production may be facilitated by a combination of this protocol with a human cellular fusion system. For efficient precursor analysis and short-term (2 months or more) monoclonal antibody production, however, the system described here may be sufficient.

Episodic LH secretion patterns in the mare during the oestrous cycle.

Jugular blood samples were obtained from 8 mares at 5- and/or 20-min intervals for 2 to 5 days during various phases of the oestrous cycle for plasma LH determination. An episodic release pattern was observed in 1 of 3 mares sampled during the ovulatory period. One mare had one secretory burst and the other mare had several periods of fluctuating plasma LH concentration. During dioestrus, episodic secretions were observed in 2 mares sampled 11 to 13 days before and, in 1 mare, 9 days after ovulation. During the 2 to 5-day period before ovulation, episodic secretion was not observed (3 mares) but plasma LH concentrations fluctuated as much as 6 ng/ml during a period of 3--4 h. Daily plasma samples were obtained form 10 mares (1--8 oestrous cycles/mare) during which 22 single, 18 double and 2 luteal-phase ovulations occurred. Dioestrous ovulations were accompanied by small increases in plasma LH (1--4 ng/ml), but many similar increases in LH were not accompanied by ovulation. No significant differences in secretory patterns were observed between single and multiple ovulations. In one mare, 4 ovulations occurred in the presence of a prolonged luteal phase; 3 were accompanied by increasing LH concentrations and the other occurred when LH was at a low concentration.